ABSTRACTThe accelerated discovery of disease-related genes emerging from genomic studies has strained the capacity of traditional genetically engineered mouse models (GEMMs) to provide in-vivo validation. Direct, somatic, genetic engineering approaches allow for accelerated and flexible genetic manipulation and represent an attractive alternative to GEMMs. In this study we investigated the feasibility, safety and efficiency of a minimally invasive, lentiviral based approach for the sustained in-vivo modification of renal tubular epithelial cells. Using ultrasound guidance, reporter vectors were directly injected into the mouse renal parenchyma. We observed transgene expression confined to the renal cortex (specifically proximal and distal tubules) and sustained beyond 2 months post injection. Furthermore, we demonstrate the ability of this methodology to induce long-term, in-vivo knockdown of candidate genes either through somatic recombination of floxed alleles or by direct delivery of specific shRNA sequences. This study demonstrates that ultrasound-guided injection of lentiviral vectors provides a safe and efficient method for the genetic manipulation of renal tubules, representing a quick and versatile alternative to GEMMs for the functional characterisation of disease-related genes.

Mentions:
To determine whether lentiviral vectors could be safely and efficiently delivered using a non-surgical approach we utilised a reporter third generation vector in which the expression of Luciferase and Strawberry fluorescent protein is driven by the constitutive EF1a promoter (ELS lentiviral vector, Fig. 1a). We performed single, low volume (10 μl) ultrasound (US) guided microinjection into the left renal parenchyma of adult (8 week, n = 5) and neonatal (7-12 day, n = 5) C57BL/6 mice (Fig. 1b). Each procedure lasted a maximum of 15 minutes and all injected mice recovered well post anaesthesia with no adverse events observed. Histopathological examination of both adult and neonatal kidneys at 7, 15 and 60 days post transduction did not reveal any morphological alterations or infiltrations within the renal parenchyma suggesting the absence of a persistent inflammatory response secondary to lentiviral infection.

Mentions:
To determine whether lentiviral vectors could be safely and efficiently delivered using a non-surgical approach we utilised a reporter third generation vector in which the expression of Luciferase and Strawberry fluorescent protein is driven by the constitutive EF1a promoter (ELS lentiviral vector, Fig. 1a). We performed single, low volume (10 μl) ultrasound (US) guided microinjection into the left renal parenchyma of adult (8 week, n = 5) and neonatal (7-12 day, n = 5) C57BL/6 mice (Fig. 1b). Each procedure lasted a maximum of 15 minutes and all injected mice recovered well post anaesthesia with no adverse events observed. Histopathological examination of both adult and neonatal kidneys at 7, 15 and 60 days post transduction did not reveal any morphological alterations or infiltrations within the renal parenchyma suggesting the absence of a persistent inflammatory response secondary to lentiviral infection.

Bottom Line:
Using ultrasound guidance, reporter vectors were directly injected into the mouse renal parenchyma.We observed transgene expression confined to the renal cortex (specifically proximal and distal tubules) and sustained beyond 2 months post injection.Furthermore, we demonstrate the ability of this methodology to induce long-term, in-vivo knockdown of candidate genes either through somatic recombination of floxed alleles or by direct delivery of specific shRNA sequences.

ABSTRACTThe accelerated discovery of disease-related genes emerging from genomic studies has strained the capacity of traditional genetically engineered mouse models (GEMMs) to provide in-vivo validation. Direct, somatic, genetic engineering approaches allow for accelerated and flexible genetic manipulation and represent an attractive alternative to GEMMs. In this study we investigated the feasibility, safety and efficiency of a minimally invasive, lentiviral based approach for the sustained in-vivo modification of renal tubular epithelial cells. Using ultrasound guidance, reporter vectors were directly injected into the mouse renal parenchyma. We observed transgene expression confined to the renal cortex (specifically proximal and distal tubules) and sustained beyond 2 months post injection. Furthermore, we demonstrate the ability of this methodology to induce long-term, in-vivo knockdown of candidate genes either through somatic recombination of floxed alleles or by direct delivery of specific shRNA sequences. This study demonstrates that ultrasound-guided injection of lentiviral vectors provides a safe and efficient method for the genetic manipulation of renal tubules, representing a quick and versatile alternative to GEMMs for the functional characterisation of disease-related genes.